Investigation of cell migration and cell fate determination is important for understanding lung development and injury/repair processes. Our long-term objective is to clarify mechanisms of early lung embryogenesis. Lung formation begins as an evagination of ventral foregut on murine gestational day 9.5 (E9.5), followed by branching morphogenesis and cell differentiation. Tracheal development in fruitflies is a simple model for identifying genes regulating formation of the respiratory system. In particular, null mutation of the Drosophila gene trachealess, encoding a PAS-domain transcription factor (TF) results in complete absence of the tracheal system. No mouse homolog of trachealess has yet been identified, but homologs of other Drosophila PAS-domain TFs are known, including single-minded (sim) and aryl hydrocarbon nuclear translocator (arnt), the latter being the homolog of Drosophila tango. These TFs are known to function as heterodimers. Recently, it was observed that mice lacking Sim2 die of respiratory failure shortly after birth. Our central hypotheses are that specific PAS-domain TFs direct mammalian lung embryogenesis; at least one of these is the functional equivalent of Trachealess; and pulmonary effects are due to downstream molecular and/or cellular events. We will address our hypotheses using four Specific Aims. AIM 1: To test the hypothesis that Sim2 plays a role in lung development. We will map spatial and temporal patterns of sim2 gene expression and carry out functional studies using antisense oligodeoxynucleotides in culture. We will analyze Sim2-null mice to determine the critical period for Sim2 function. AIM 2: To test the hypothesis that other PAS-domain TFs (953 and Arnt) regulate lung development through heterodimer formation using a genetic approach, which reflects biochemical interactions. Our novel mouse cDNA 953, approximately 50 percent identical to trachealess, is expressed in embryonic lung. Heterodimer formation with Arnt/Tango is required for function of both Sim2 and trachealess. Interaction of these Tfs will be tested in vivo by interbreeding hemizygous knockout mice. AIM 3: To test the hypothesis that pulmonary defects occurring in mice deficient for specific PAS-domain TFs are due to aberrant downstream molecular and/or cell differentiation. This investigation will open new avenues to understanding of mechanisms of lung embryogenesis and injury/repair processes.